JPWO2006054755A1 - Method for screening drug candidate substance - Google Patents

Abstract

A method for screening a drug candidate mainly metabolized by the drug metabolizing enzymes CYP2D6, CYP2C9, and CYP2C19 using a chimeric mouse transplanted with human hepatocytes, wherein the drug candidate substance is CYP2D6, CYP2C9, or CYP2C19 deficient A method for determining whether or not it is metabolized is provided. Also provided is a method for determining whether a drug candidate induces or inhibits the activity of any one of the drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 or CYP3A4 using a human liver transplanted chimeric mouse.

Description

  The invention of this application relates to a method for screening drug candidates that are mainly metabolized by the drug metabolizing enzymes CYP2D6, CYP2C9, or CYP2C19. The present invention also relates to a method for determining which of the CYP group drug metabolizing enzymes the drug candidate substance induces or inhibits its activity.

The stage of selecting drug candidates in drug development is of greatest interest to pharmaceutical companies. It is common knowledge that pharmaceutical development requires a huge R & D cost of more than 30 billion yen and more than 10 years. Therefore, it must be avoided that the drug candidate substance is canceled during the course of research and development. Recently, domestic pharmaceutical companies have been developing Japan, the United States and Europe at the same time. Racial and ethnic differences in the effectiveness have hindered simultaneous development, and companies have avoided the development of drug candidate substances that have racial differences. This is the current situation. In general, racial differences in drug metabolizing enzymes are considered as factors causing racial differences in pharmacokinetics. In particular, genetic polymorphisms of drug metabolizing enzymes Cytochrome P450 (CYP) 2D6, CYP2C9, or CYP2C19 are mainly used in these enzymes. It is thought to cause racial differences in the pharmacokinetics of metabolized drug candidates. For example, about 10% of CYP2D6 is Western and there are deficient patients (PM: Poor Metabolizer). Currently, compounds mainly metabolized by CYP2D6 are not selected as drug candidate substances.
In addition, the drug interaction of pharmaceuticals is an important problem in the medical field, such as causing serious symptoms depending on the combination of drugs. In the event of drug interaction problems, pharmaceutical companies may develop a situation where drugs are withdrawn from the market. Therefore, drug interactions based on the induction and inhibition of drug-metabolizing enzymes are important decision-making factors in pharmaceutical research and development, so prediction of drug interactions in humans is an important research and development concern. ing.
The inventors of this application have succeeded in creating a chimeric mouse in which human hepatocytes involved in human drug metabolism are substituted in a high proportion of mouse hepatocytes in the mouse liver, and have filed a patent application ( Patent Documents 1 and 2).
Japanese Patent Publication No. 2002-45087 International Patent Publication WO2003 / 080821 Pamphlet

However, it is difficult to determine whether a drug candidate substance is mainly metabolized by CYP2D6, CYP2C9, or CYP2C19 at a stage where there is no radiolabeled label compound. When an ADME test is performed using a label compound of a drug candidate substance, the overall metabolic pathway is revealed, and it can be understood how much the metabolic process involving CYP2D6, CYP2C9, or CYP2C19 occupies the metabolism of the drug candidate substance. Therefore, in the situation where there is no label compound in the search stage, it is not possible to accurately estimate the involvement of CYP2D6, CYP2C9, or CYP2C19 metabolism in the drug candidate substance.
The invention of this application has been made in view of the circumstances as described above, and when the drug candidate substance is a substance mainly metabolized by CYP2D6, CYP2C9, or CYP2C19, the genetic polymorphism is present in the pharmacokinetics. It is an object to provide a screening method for determining whether or not it occurs.
To date, there has been no in vivo system for detecting human drug interactions. In the in vitro system, the drug exposure concentration to the cells is always in a high state, so it is difficult to predict the drug interaction in the human body. Therefore, an object of the invention of this application is to provide a screening method for detecting a human drug interaction in vivo.
In this application, as a first invention for solving the above-mentioned problem, whether a drug candidate substance mainly metabolized by the drug metabolizing enzyme CYP2D6, CYP2C9, or CYP2C19 is metabolized by a CYP2D6, CYP2C9, or CYP2C19 deficient person. A method for determining whether or not
(1) A step of administering a drug candidate substance to each of a highly substituted chimeric mouse in which transplanted human hepatocytes occupy 70% or more of the mouse liver and a low substituted chimeric mouse in which almost no human hepatocytes are engrafted. ;
(2) a step of measuring the concentration of a drug candidate substance in serum collected from each of a high-substituted chimeric mouse and a low-substituted chimeric mouse over time;
And a drug candidate substance that is metabolized significantly faster in a highly substituted chimeric mouse than a low substituted chimeric mouse, is determined to be a substance that is not metabolized in a CYP2D6, CYP2C9, or CYP2C19 deficient person I will provide a.
In addition, as a second invention, this application is a method for determining whether or not a drug candidate substance promotes or inhibits the activity of a drug metabolizing enzyme,
(1) a step of administering a drug candidate substance to a highly substituted chimeric mouse;
(2) administering a plurality of indicator compounds metabolized by each of a plurality of drug metabolizing enzymes to a highly substituted chimeric mouse;
(3) A step of measuring the concentration of the indicator compound in the serum collected from the highly substituted chimeric mouse over time;
To determine which drug metabolizing enzyme induces or inhibits the activity of the drug candidate substance by comparing with the concentration of the indicator compound in the serum when the drug candidate substance is not administered A method is provided.

FIG. 1 is a diagram showing an example of a detected peak obtained by analyzing a mouse serum sample to which 5 compounds have been simultaneously administered by LC-MS / MS.
FIG. 2 is a graph showing changes over time in the caffeine concentration detected in mouse serum when caffeine was administered to mice. Chimeric mice transplanted with donor cells (IVT079) were used.
FIG. 3 is a graph showing the change over time in the concentration of tolbutamide detected in mouse serum when tolbutamide was administered to mice. Chimeric mice transplanted with donor cells (IVT079) were used.
FIG. 4 is a diagram showing the change over time of the concentration of omeprazole detected in mouse serum when omeprazole was administered to mice. Chimeric mice transplanted with donor cells (IVT079) were used.
FIG. 5 is a graph showing the change over time in the concentration of dextromethorphan detected in mouse plasma when dextromethorphan is administered to mice. Chimeric mice transplanted with donor cells (IVT079) were used.
FIG. 6 is a graph showing the change over time of the erythromycin concentration detected in mouse plasma when erythromycin was administered to mice. Chimeric mice transplanted with donor cells (IVT079) were used.
FIG. 7 is a graph showing changes over time in the concentration of caffeine or tolbutamide detected in mouse plasma when caffeine or tolbutamide was administered to mice. Chimeric mice transplanted with donor cells (BD51) were used.
FIG. 8 is a graph showing the change over time in the concentration of omeprazole or dextromethorphan detected in mouse plasma when omeprazole or dextromethorphan was administered to mice. Chimeric mice transplanted with donor cells (BD51) were used.
FIG. 9 is a graph showing changes over time in the erythromycin concentration detected in mouse plasma when erythromycin was administered to mice. Chimeric mice transplanted with donor cells (BD51) were used.
FIG. 10 shows the change over time in the concentration of each administered substance detected in the plasma of highly substituted chimeric mice when caffeine, tolbutamide, omeprazole, dextromethorphan, and erythromycin were administered to mice without or after administration of paroxetine. FIG. Chimeric mice transplanted with donor cells (BD51) were used.

The method of the first invention is:
(1): a step of administering a drug candidate substance to each of a highly substituted chimeric mouse and a low substituted chimeric mouse;
(2): a step of measuring the concentration of a drug candidate substance in serum collected from each of a high-substituted chimeric mouse and a low-substituted chimeric mouse over time;
including. And determining that the drug candidate substance that is metabolized significantly faster in the high substitution chimeric mouse than in the low substitution chimera mouse is a substance that is not metabolized in a CYP2D6, CYP2C9, or CYP2C19 deficient person. is there.
A highly substituted chimeric mouse is a mouse in which transplanted human hepatocytes occupy 70% or more of the mouse liver, and can be obtained by the method described in Patent Document 1 or 2. In particular, the method of Patent Document 2 (a method of rearing a mouse in a state protected from the attack of human complement produced by transplanted human hepatocytes) is preferable as a method of achieving a human hepatocyte replacement rate of 70% or more. . The low-replacement chimeric mouse is a mouse in which human hepatocytes are hardly established (specifically, the replacement ratio of human hepatocytes is less than 1%).
These chimeric mice are used for administration of drug candidate substances, for example, about 60 days after transplantation of human hepatocytes. The human hepatocyte replacement rate of the chimeric mouse can be confirmed in advance by a method such as measurement of the human albumin concentration in the mouse blood. Furthermore, a more accurate replacement rate can be confirmed by performing anatomical examination of the liver of the chimeric mouse after the test by the method described in Patent Document 2 and the like.
A drug candidate substance is a substance in the process of drug development and is a substance that requires prediction of drug interaction in humans. Such a substance may be a main component for the medicinal effect of a pharmaceutical or a composition containing the main component. The dosage of the drug candidate substance varies depending on the type of the disease targeted by the substance, the type of substance composition, the administration route, etc., but can be about 0.1 mg / kg body weight to 2000 mg / kg body weight. The route of administration can be oral, subcutaneous, intravenous or intraperitoneal administration depending on the type of drug candidate substance and its suitable dosage form.
The degree of metabolism of the drug candidate substance in the liver of the chimeric mouse can be determined by the standard method in the technical field (for example, the chromatographic method described in the Examples, etc.) with respect to the concentration of the drug candidate substance in mouse serum. The measurement can be performed over time (approximately 15 minutes to 24 hours) from 30 minutes to 24 hours after administration of the candidate substance.
Depending on the concentration of the drug candidate substance in the serum (the concentration of the substance metabolized by the drug metabolizing enzyme in the liver), the drug candidate substance is a substance that is easily metabolized by a CYP2D6, CYP2C9, or CYP2C19 deficient person, or a substance that is difficult to metabolize. Determine whether. Specifically, when a drug candidate substance in serum is metabolized significantly faster in a high substitution chimeric mouse than in a low substitution chimera mouse, the drug candidate substance is considered to be metabolized by CYP2D6, CYP2C9, or CYP2C19. It is done. Therefore, it is determined that this drug candidate substance is a substance that is not metabolized by a CYP2D6, CYP2C9, or CYP2C19 deficient person.
“Significantly metabolized” means that the AUC (Area under the curve) calculated from the concentration of the drug candidate in mouse blood is about 2/3, preferably Means an average AUC value of about 1/2 to 1/3 compared to low-substituted mice.
The method of the second invention is
(1): a step of administering a drug candidate substance to a highly substituted chimeric mouse;
(2): a step of administering a plurality of indicator compounds metabolized by each of a plurality of drug metabolizing enzymes to a highly substituted chimeric mouse; and (3): a concentration of the indicator compound in serum collected from the highly substituted chimeric mouse. Measuring over time;
including. Then, by comparing with the concentration of the indicator compound in the serum when the drug candidate substance is not administered, it is possible to determine which drug metabolizing enzyme induces or inhibits the activity of the drug candidate substance. is there.
Human drug-metabolizing enzymes are CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C10, CYP2C18, CYP2C3, CYP2A3, CYP2C3, CYP2E3, CYP2E3 For example, the indicator compounds metabolized by CYP1A2 are caffeine, CYP2C9 is tolbutamide, CYP2D6 is dextromethorphan, CYP2C19 is omeprazole, CYP3A4 is erythromycin, and the like. Substance that promotes the activity of each enzyme by administering a drug candidate substance to a highly substituted chimeric mouse, then administering a mixture of these indicator compounds, and measuring the serum concentration of each compound over time It is possible to determine whether the substance is an inhibitory substance. For example, if caffeine metabolism is promoted in the mixture of indicator compounds and the serum concentration is decreased, the drug candidate substance can be determined to be a substance that specifically promotes the activity of CYP1A2. .
Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples.

以上の結果から、高置換キメラマウスと低置換キメラマウスにおける各種指標薬物のファーマコキネティクスを比較すると、２Ｃ１９と３Ａ４の指標薬物を投与した場合には顕著な差は認められなかったが、移植に用いたドナー肝細胞によっては、ＣＹＰ１Ａ２，ＣＹＰ２Ｃ９，およびＣＹＰ２Ｄ６の指標薬物を投与した場合、高置換キメラマウスに比べて、低置換キメラマウスで代謝速度が遅いことが判明した。すなわち、ＣＹＰ１Ａ２，ＣＹＰ２Ｃ９，またはＣＹＰ２Ｄ６に関して、低置換キメラマウスはヒトでのＰＭ（Ｐｏｏｒ Ｍｅｔａｂｏｌｉｚｅｒ）、高置換キメラマウスは代謝速度が速いＥＭ（Ｅｘｔｅｎｓｉｖｅ Ｍｅｔａｂｏｌｉｚｅｒ）に相当することが明らかになった。これにより、医薬品開発において、ＣＹＰ２Ｃ９、またはＣＹＰ２Ｄ６で主として代謝される医薬候補物質の遺伝多型における薬物動態の違いを判定できる。また、移植に用いるドナー肝細胞を選択することにより、ＣＹＰ２Ｃ１９で主として代謝される医薬候補物質の遺伝多型における薬物動態の違いも判定できると考えられた。
ＣＹＰ２Ｃ９、ＣＹＰ２Ｃ１９、またはＣＹＰ２Ｄ６で主として代謝される医薬候補物質の高置換および低置換キメラマウスにおけるファーマコキネティックスを調べることにより、医薬候補物質の開発継続かあるいは中止かの意思決定をすることが可能となる。Pharmacokinetic Test 1 Using Chimeric Mouse Material and Method 1-1 Test Substance Caffeine (CYP1A2), a standard drug of cytochrome P450 (CYP), was used to evaluate pharmacokinetics in human hepatocyte chimeric mice using an index drug. ), Tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and erythromycin (CYP3A4).
1-2 Animals Used Chimera mice produced at Hiroshima Prefectural Institute of Industrial Science and Technology or Phoenix Bio Inc. were used. That is, human hepatocytes (purchased from IVT079, In Vitro Tcnology, or purchased from BD51, BD Genest) were transplanted into the spleen of uPA (+ / +) / SCID mice, which are mice with liver damage and immunodeficiency properties. Then, a mouse in which human hepatocytes were engrafted in the mouse liver and proliferated was prepared. Mice in which the mouse liver was replaced with human hepatocytes by 70% or more (highly substituted chimeric mice) and low-substituted chimeric mice in which human hepatocytes were transplanted but the substitution rate was almost 0%, or non-transplanted mice (zero-substituted mice) Three of each were used. In addition, three uPA (− / −) / SCID mice were used as controls.
Each mouse was allowed to freely receive water (containing 0.012% hypochlorous acid) and a sterilized solid feed containing vitamin C (CRF-1, oriental yeast), and was bred at room temperature of 24 ± 2 ° C. The highly substituted chimeric mice were intraperitoneally administered nafamostat mesylate injection (0.3 mg / 0.2 ml / body) twice a day.
1-3 Preparation of Administered Drug Each drug was prepared at 5 times the administration concentration, and 5 compounds were mixed immediately before administration so that the final concentration would be the respective administration concentration. The administration concentration of caffeine, which is a standard drug of CYP1A2, is 25 mg / kg b. w. 12.5 mg was weighed and suspended in 1 ml of CMC solution in an agate mortar to prepare a 12.5 mg / ml suspension of 5 times the administration concentration. The dose concentration of tolbutamide, the standard drug for CYP2C9, was 8.35 mg / kg b. w. 4. 175 mg was weighed and suspended in 1 ml of CMC solution in an agate mortar to prepare a 4.175 mg / ml suspension having a dose concentration 5 times higher. The dose concentration of dextromethorphan, the standard drug for CYP2D6, is 2.5 mg / kg b. w. 20 mg of dextromethorphan hydromide monohydrate was weighed and suspended in 16 ml of CMC solution in an agate mortar to prepare a 1.25 mg / ml suspension of 5 times the administration concentration. The administration concentration of omeprazole, the standard drug for CYP2C19, was 8.35 mg / kg b. w. 20 mg of 1 vial was dissolved in 4.76 ml of milli-Q water to prepare a 4.2 mg / ml aqueous solution having a dose concentration 5 times. The administration concentration of erythromycin, the standard drug for CYP3A4, was 125 mg / kg b. w. A 500 mg erythromycin lactobionate vial was dissolved in 8 ml of milli-Q water to prepare an aqueous solution of 62.5 mg / ml, 5 times the dose concentration. 1 ml of each prepared drug is mixed to 5 ml, and the suspension is 10 μl / g b. w was administered to mice.
1-4 Administration Method and Serum Collection Method Three compounds each of high substitution chimera mouse, low substitution chimera mouse (or zero substitution mouse), uPA (− / −) / SCID mouse are forcibly orally administered or administered intravenously at the same time. Went. Blood samples were collected at 0.25 (only intravenous administration), 0.5, 1, 2, 4 (only oral administration) after administration, 15 μl each from the tail vein of the mouse 8 hours later, and further centrifuged at 3,500 rpm for 5 minutes Later, serum (supernatant) was collected and stored in a freezer at −30 ° C.
1-5 Quantitative Analysis and Analysis of Serum Drug Plasma samples were transported to Sumika Analysis Center, Inc. in dry ice, and measured by LC-MS / MS by the following method.
1-5-1 Measurement target substance standard solution and I.I. S. Preparation of standard solution Caffeine, tolbutamide, omeprazole, dextromethorphan hydrobromide monohydrate, erythromycin lactobionate were diluted with methanol, 2000, 1000, 500, 100, 50, 10, 5, 1, 0.5, A standard solution of 0.1 ng / ml was prepared. Dissolve phenytoin in methanol to prepare a standard solution of 10,1 mg / ml. S. A standard solution was used.
1-5-2 Measuring equipment and measuring conditions (LC-MS / MS)
The liquid chromatograph was SIL-HTC and LC-10A series (Shimadzu Corporation), and the column was Synergi 4μ Polar-RP 80A, 150 mm L.P. x 2.00 mmI. D. , 4 μm (Phenomenex) was used at 30 ° C. Methanol / 10 mmol / l ammonium acetate solution / acetic acid (700: 300: 2, v / v / v) was used as the mobile phase, and the flow rate was 0.2 ml / min. The injection amount was 2 μl, the measurement time was 10 min, and the set temperature of the autosampler was 4 ° C. As the mass spectrometer, a Tandem Mass Spectrometer API 4000 (Applied Biosystems / MDS SCIEX) was used. The API interface was Turbo V (ESI probe), and the ionization mode was the positive ion detection mode. The detection product of Detection MRM is caffeine: m / z 195.1 → m / z 138.2 (170 msec / l scan), tolbutamide: m / z 271.1 → m / z 74.3 (170 msec / l scan), omeprazole : M / z 346.1 → m / z 198.2 (150 msec / l scan), dextromethorphan: m / z 272.2 → m / z 215.3 (150 msec / l scan), erythromycin: m / z 734.4 → m /Z158.4 (150 msec / l scan), phenytoin: m / z 253.1 → m / z 182.3 (150 msec / l scan).
1-5-3 Preparation of added calibration curve sample Weigh 5 μl of control mouse serum into an Eppendorf tube, add 100 μl of the standard solution for the substance to be measured (mixture of 5 compounds), final concentration 0.1, 0.5, 1 5, 10, 50, 100, 500, 1000 ng / ml. Next, 1 μg / ml of I.V. S. 10 μl of the standard solution was added and mixed well by sonication and vortex mixer. This was centrifuged (15,000 rpm, 5 min, room temperature), and then the supernatant was subjected to centrifugal filtration (10,000 rpm, 2 min, room temperature) with a filtration filter to obtain an LC-MS / MS injection sample.
1-5-4 Preparation of Measurement Sample 100 μl of methanol was added to an Eppendorf tube containing 5 μl of the measurement sample (plasma), and 1 μg / ml of I.D. S. After adding 10 μl of the standard solution, it was thoroughly mixed by sonication and vortex mixer. This was centrifuged (15,000 rpm, 5 min, room temperature), and then the supernatant was subjected to centrifugal filtration (10,000 rpm, 2 min, room temperature) with a filtration filter to obtain an LC-MS / MS injection sample.
2 Test Results The quantitative value of the plasma drug concentration was determined from the peak area ratio obtained by LS-MS / MS measurement of the added calibration curve sample. FIG. 1 shows an example of peaks obtained by LS-MS / MS measurement. The relationship between the blood collection time and the concentration of the administered drug in the blood was graphed from the quantitative value (FIG. 2-9). Analyzing the maximum plasma concentration (Cmax), half-life (t1 / 2), area under the blood concentration curve (AUC), bioavailability, and clearance from the graph, predicting human pharmacokinetics Went. The results are shown in Tables 1, 2 and 3.
The AUC involving almost all CYPs was lower in the highly substituted chimeric mice compared to the low substituted chimeric mice. In chimeric mice using IVT079 for donor hepatocytes, the AUC when tolbutamide (CYP2C9) and dextromethorphan (CYP2D6) were administered was significantly lower in the high substitution rate chimeric mice than in the low substitution rate chimeric mice . Similar results were obtained for the bioavailability. In chimeric mice using BD51 for donor hepatocytes, AUC when caffeine (CYP1A2) and tolbutamide (CYP2C9) were administered was significantly lower in high substitution rate chimeric mice than in low substitution rate chimeric mice.

From the above results, when comparing the pharmacokinetics of various index drugs in high substitution chimera mice and low substitution chimera mice, no significant difference was observed when 2C19 and 3A4 index drugs were administered. Depending on the donor hepatocytes used, it was found that when the index drugs CYP1A2, CYP2C9, and CYP2D6 were administered, the metabolic rate was low in the low substitution chimeric mice compared to the high substitution chimeric mice. That is, with regard to CYP1A2, CYP2C9, or CYP2D6, it has been clarified that a low-substituted chimeric mouse corresponds to human PM (Poor Metabolizer) and a high-substituted chimeric mouse corresponds to EM (Extensible Metabolizer) having a high metabolic rate. Thereby, in drug development, the difference in the pharmacokinetics in the genetic polymorphism of the drug candidate substance mainly metabolized by CYP2C9 or CYP2D6 can be determined. Moreover, it was thought that the difference in the pharmacokinetics in the genetic polymorphism of the drug candidate substance mainly metabolized by CYP2C19 could be determined by selecting the donor hepatocytes used for transplantation.
By investigating pharmacokinetics in high-substitution and low-substitution chimera mice with drug candidates mainly metabolized by CYP2C9, CYP2C19, or CYP2D6, it is possible to decide whether to continue or stop the development of drug candidates It becomes.

以上の結果から、高置換キメラマウスに被検物質を投与し各指標物質のＡＵＣの変化を調べることにより、被検物質の誘導または阻害活性を調べることが可能である。したがって、キメラマウスを用いることにより、ヒトにおける薬物相互作用を予測することが可能である。Pharmacokinetics test in CYP2D6 inhibition using chimeric mice 1 Materials and methods 1-1 Evaluation of pharmacokinetics in human hepatocyte chimeric mice using CYP2D6 enzyme with test substance (CYP2D6 enzyme inhibitor) using indicator drug For this purpose, five compounds of caffeine (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), and erythromycin (CYP3A4), which are index drugs of cytochrome P450 (CYP), were used.
1-2 Animals Used Chimera mice produced by Phoenix Bio Inc. were used. That is, human hepatocytes (BD51, purchased from BD Genist) were transplanted into the spleen of uPA (+ / +) / SCID mice, which are mice with liver damage and immunodeficiency properties, and human hepatocytes live in the mouse liver. Three mice that had arrived and proliferated were prepared.
Each mouse ingested and raised water and feed as in Example 1. In addition, to the highly substituted chimeric mice, nafamostat mesylate injection solution (0.3 mg / 0.2 ml / body) was intraperitoneally administered twice a day as in Example 1.
1-3 Preparation of Inhibitory Drug Paroxetine was dissolved in milli-Q water (using bath-type ultrasound) to prepare 6 mg / ml. The prepared drug was 5 μl / g b. w. (30 mg / kg bw / day) was administered intraperitoneally to mice.
1-4 Preparation of index drug The same procedure as in Example 1 was performed.
1-5 Administration Method and Plasma Collection Method Paroxetine was intraperitoneally administered to highly substituted chimeric mice for 3 days. Thereafter, five compounds were simultaneously orally administered as in Example 1 and blood was collected over time, and plasma was stored.
1-6 Quantification and analysis of drug in plasma The same procedure as in Example 1 was performed.
1-6-1 Measurement target substance standard solution and I.I. S. Preparation of standard solution The same procedure as in Example 1 was performed.
1-6-2 Measuring equipment and measuring conditions (LC-MS / MS)
The same operation as in Example 1 was performed.
1-6-3 Preparation of additive calibration curve sample The same procedure as in Example 1 was performed.
1-6-4 Preparation of measurement sample The same procedure as in Example 1 was performed.
2 Test Results The quantitative value of the plasma drug concentration was determined from the peak area ratio obtained by LS-MS / MS measurement of the added calibration curve sample. The relationship between the blood collection time and the concentration of the administered drug in the blood was graphed from the quantitative value (FIG. 10). Cmax, t1 / 2, and AUC were analyzed from the graph.
The results are shown in Table 4. Inhibition of metabolic activity of CYP1A2, 2C19, 2D6, 3A4 was observed by administration of paroxetine. The metabolic activity of CYP2D6, which is said to be specifically inhibited by paroxetine, was particularly inhibited.

From the above results, it is possible to examine the induction or inhibition activity of the test substance by administering the test substance to the highly substituted chimeric mouse and examining the change in AUC of each indicator substance. Therefore, it is possible to predict drug interactions in humans by using chimeric mice.

Claims (2)

A method for determining whether or not a drug candidate substance that is mainly metabolized by a drug metabolizing enzyme CYP2D6, CYP2C9, or CYP2C19 is metabolized by a CYP2D6, CYP2C9, or CYP2C19 deficient person,
(1) A step of administering a drug candidate substance to each of a highly substituted chimeric mouse in which transplanted human hepatocytes occupy 70% or more of the mouse liver and a low substituted chimeric mouse in which almost no human hepatocytes are engrafted. ;
(2) a step of measuring the concentration of a drug candidate substance in serum collected from each of a high-substituted chimeric mouse and a low-substituted chimeric mouse over time;
And a drug candidate substance that is metabolized significantly faster in a highly substituted chimeric mouse than a low substituted chimeric mouse, is determined to be a substance that is not metabolized in a CYP2D6, CYP2C9, or CYP2C19 deficient person . A method for determining whether a drug candidate substance promotes or inhibits the activity of a drug metabolizing enzyme, comprising:
(1) a step of administering a drug candidate substance to a highly substituted chimeric mouse;
(2) administering a plurality of indicator compounds metabolized by each of a plurality of drug metabolizing enzymes to a highly substituted chimeric mouse;
(3) A step of measuring the concentration of the indicator compound in the serum collected from the highly substituted chimeric mouse over time;
To determine which drug metabolizing enzyme induces or inhibits the activity of the drug candidate substance by comparing with the concentration of the indicator compound in the serum when the drug candidate substance is not administered A method characterized by.

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